Structural biology of voltage-gated calcium channels

ABSTRACT Voltage-gated calcium (Cav) channels mediate Ca2+ influx in response to membrane depolarization, playing critical roles in diverse physiological processes. Dysfunction or aberrant regulation of Cav channels can lead to life-threatening consequences. Cav-targeting drugs have been clinically used to treat cardiovascular and neuronal disorders for several decades. This review aims to provide an account of recent developments in the structural dissection of Cav channels. High-resolution structures have significantly advanced our understanding of the working and disease mechanisms of Cav channels, shed light on the molecular basis for their modulation, and elucidated the modes of actions (MOAs) of representative drugs and toxins. The progress in structural studies of Cav channels lays the foundation for future drug discovery efforts targeting Cav channelopathies.


Introduction
Ca v channels constitute a group of integral membrane proteins that facilitate the selective influx of Ca 2+ , a second messenger involved in numerous cellular events, into the cytosol upon membrane depolarization.These proteins exhibit diverse tissue distributions and play pivotal roles in a wide array of physiological processes, including muscle contraction, neurotransmitter release, hormone secretion, and cell death [1,2].The history of Ca v channel discovery has been comprehensively reviewed by Tsien, Barrett and Dolphin [3,4].In mammals, the ten primary subtypes of Ca v channels are classified into three subfamilies, Ca v 1 (Ca v 1.1-Ca v 1.4), Ca v 2 (Ca v 2.1-Ca v 2.3) and Ca v 3 (Ca v 3.1-Ca v 3.3), based on sequence similarities of the α1 subunit (Figure 1a) [5][6][7][8][9][10].As implied by the family name, the open probability of Ca v channels is regulated by membrane potential.Ca v 1 and Ca v 2 are recognized as high-voltage activated Ca 2+ (HVA) channels, given their activation at more depolarized membrane potential [1].In contrast, Ca v 3 channels form a subset of low-voltage activated Ca 2+ channels (LVA) that can be activated at depolarization only slightly above the resting membrane potential and, in some cases, may even require conditioning hyperpolarization for priming [11,12].
Dysfunction of Ca v channels is implicated in a range of disorders, including cardiac arrhythmias, primary aldosteronism, ataxia, migraine, cognitive anomalies, and autism.The physiology, pathology, and pharmacology of Ca v channels have been extensively documented by Zamponi, Dolphin, et al [2,[13][14][15].For instance, small molecule drugs, e.g.1,4-dihydropyridines (DHP, e.g.nifedipine), benzothiazepines (e.g.diltiazem) and phenylalkylamines (e.g.verapamil), that inhibit the cardiovascular L-type Ca v channels (LTCCs), have been widely used in clinical practice for decades to treat hypertension and cardiac arrhythmias (Figure 1a,b) [16,17].Simultaneously, peptide toxins from spiders, snakes, and marine creatures, which selectively bind to specific regions of ion channels, have historically been employed to investigate the subtype-specific inhibitory effects [18,19].Achieving a precise understanding of the modes of actions (MOAs) of Ca vtargeting drugs requires the structural elucidation of drug-bound channels, a daunting challenge that was finally overcome thanks to the resolution revolution Please refer to table 1 for PDB codes and additional details.(c) the HVA channels consist of at least three subunits: the transmembrane α1 subunit, the extracellular α2δ subunit, and the cytosolic β subunit.The α1 subunit comprises four homologous of single particle cryo-electron microscopy (cryo-EM).
The pore-forming α1 subunits of Ca v channels exhibit nearly identical topologies, with approximately 2000 residues arranged into four homologous repeats (designated I-IV), each containing six transmembrane helices (S1-S6) (Figure 1c).The S5 and S6 segments from all four repeats collectively enclose the central pore domain (PD), forming a specific pathway for the selective passage of Ca 2 + ions across the cell membrane.Meanwhile, the S1-S4 segments within each repeat constitute the voltage-sensing domain (VSD), where the S4 segment carries repetitive positively charged residues (arginine or lysine) as gating charges.The four sets of VSDs encircle PD in a domain-swapped manner, cooperatively coupling the fluctuations in membrane potential to the pore gating of ion conduction [5,[20][21][22][23].
The core α1 subunit is self-sufficient for the autonomous function of Ca v 3 channels, whereas the Ca v 1 and Ca v 2 subfamilies necessitate auxiliary extracellular α2δ and intracellular β subunits for proper membrane trafficking and physiological functions (Figure 1c) [11,[24][25][26].The Ca v 1.1 channels, specialized for skeletal muscle, also associate with a transmembrane γ subunit sharing the same folding pattern with claudins [27,28].Within the Ca v 1 and Ca v 2 subfamilies, the α1 subunits interact with the α2δ subunits through the extracellular segments, while interacting with the β subunits on the cytosolic side (Figure 1c).Four mammalian α2δ genes, namely CACNA2D1 to D4, encode the extracellular subunits α2δ-1 to −4, respectively [29].These gene products are initially the preproteins for the α2δ subunits, which will undergo post-translational proteolysis into α2 and δ proteins as the mature forms.Nonetheless, these two proteins remain interconnected due to inter-subunit disulfide bond formation before proteolytic cleavage [30][31][32][33].The mature α2δ subunits are highly glycosylated proteins that aid in trafficking the α1 subunits to the cell membrane, enhancing channel expression, and modulating channel properties [26].In addition, four subtypes of β subunits, β1-β4 with multiple splice isoforms, contribute to channel trafficking, modulate gating properties, and interact with intracellular signaling molecules [24,[34][35][36][37][38].The specific assortment of subunit constituents varies depending on the particular type of Ca v channel and the specific tissue or cell type in which it is located.
Obtaining accurate 3D structures for Ca v channels lays the foundation for unveiling the working mechanisms and advancing drug discovery endeavors.The first cryo-EM structure of Ca v channels was reported by our group in 2015 [28].Following that, various subtypes of Ca v channels alone and in complex with small molecule and peptide ligands have been extensively characterized (Table 1).This review will provide a comprehensive overview of recent advancements in the structural architecture and pharmacology of Ca v channels, aiming to facilitate future physiological investigations and the development of innovative drugs.

Structural architecture of Ca v channels
Ca v channels were first purified from the T-tubule membranes of rabbit skeletal muscle and later from the cardiac and brain membranes [53][54][55][56][57][58].The endogenously purified rabbit Ca v 1.1 channel has historically served as the prototype for the structural analysis of working and drug modulation mechanisms [27,28,39,40].More recently, the establishment of a heterologous expression system for Ca v channels has enabled the generation of sufficient samples encompassing diverse subtypes, repeats (I-IV), each containing six helices (S1-S6).The S1-S4 helices of each repeat constitute the voltage-sensing domain (VSD), and four sets of S5 and S6 helices form the ion-conducting pore domain (PD).The S4 segments, carrying repetitive positively charged residues, are highlighted as yellow cylinders.The auxiliary α2δ subunit is composed of two polypeptides (α2 and δ) associated by an inter-subunit disulphide bond.The glycosyl phosphatidylinositol (GPI) group at the C-terminus of δ anchors it to the outer leaflet of the cell membrane.The α2δ subunit contains one von willebrand factor-A (VWA) domain and four cache domains.The two conserved domains of the β subunit, src homology 3 (SH3) and a guanylate kinase-like domain (GK), are also shown.GK interacts with the α1-interacting domain (AID) motif located at the intracellular loop between repeat I and II.
facilitating the structural elucidation of a broad range of Ca v channels [44].In this section, we will delve into a comprehensive review of the precise structural architectures of representative members within each Ca v subfamily.

Subunit architecture of Ca v channels
The endogenously purified rabbit Ca v 1.1 and recombinantly expressed Ca v 2.2 channels both comprise a transmembrane α1 subunit, along with auxiliary α2δ-1 and β subunits.The rabbit Ca v 1.1 was also recognized for its interaction with the γ subunit (Figure 2a, left).Examining the overall structures of representative members within the three Ca v subfamilies provides detailed insight into their subunit architectures (Figure 2a).The extracellular α2δ-1 subunit comprises a single von Willebrand factor-A (VWA) domain and four tandem Cache domains (Cache1 to 4), which are intertwined in the primary sequence (Figure 2b) [27,28].Despite undergoing posttranslational proteolysis, the α2δ-1 subunit  loop) (Figure 2a,b).Furthermore, the interface between VWA and α1 subunit involves the coordination of a Ca 2+ ion, facilitated by the metal iondependent adhesion site (MIDAS) motif and the Asp residue on the S1-2 I loop of the α1 subunit.
The cytosolic β subunits have been identified to interact with the α1-interacting domain (AID), an 18-aa motif at the proximal region of the intracellular loop between domain I and II [59].All β subunits feature a conserved src homology 3 (SH3) and a guanylate kinase-like domain (GK), where the AID motif binds to the deep groove on the GK domain (Figure 2a).Besides the wellcharacterized interface, the β3 subunit was recognized for its additional interactions with the extended cytosolic segments of S6 II (S6 II C) in the Ca v 2.2 structure (Figure 2a, middle) [46].
In the Ca v 1.1 structure, the γ subunit, which shares the same fold as claudins, interacts with VSD IV through both transmembrane segments and cytosolic loops.The physical contacts between the γ subunit and the α1 subunit may potentially impact the conformational alterations of VSD IV during voltage-dependent activation or inactivation.The interactions provide the molecular basis for the diverse modulation effects, including antagonistic influences, exerted by the γ subunit on channel properties (Figure 2a, left) [60,61].

Diverse PD conformations in the structures of Ca v channels
Four sets of transmembrane S5 and S6 along with the supporting helices P1 and P2 collectively form the central PD, serving as the high-throughput and highly selective pathway for the influx of Ca 2+ ions.Inquiries into the precise mechanisms by which Ca v channels specifically bind to Ca 2+ ions and swiftly release them have held substantial importance for decades [21,[62][63][64].The high-resolution 3D structures provide the opportunity to reassess previous biophysical measurements, advancing our understanding of the selectivity and permeation of Ca v channels.
In the resolved structures, the EEEE/EEDD side chains protrude into the pore lumen, constituting the intrachannel binding site for Ca 2+ ion (Figure 3a).The EM densities within the selectivity filter (SF) vestibule can be deconvoluted as two closely spaced Ca 2+ ions: one situated directly at the EEEE/ EEDD locus, and the other positioned in proximity to the carbonyl groups of the − 1 residues at a Ca 2+ concentration of 10 mM (Figure 3a, left and middle).Moreover, the EEEE locus is coordinated with a single Ca 2+ ion in the presence of 0.5 mM Ca 2+ (Figure 3a, right).It was postulated that Ca v channel comprised an intrapore Ca 2+ binding site of low affinity (K 1/2 ~10 mM), and an external binding site of high affinity (K 1/2 ~0.3 μM) [65].The presence of intrapore Ca 2+ density at sub-millimolar Ca 2+ concentrations supersedes the initial model of the low-affinity binding site within pore.Nonetheless, mutational studies have failed to reveal an additional high-affinity Ca v binding site other than the one formed by EEEE residues [66,67].Structural and mutational analyses suggest that the EEEE motif tightly binds a single Ca 2+ to impede Na + influx at low concentrations, while undergoing spatial rearrangements to permit Ca 2+ influx as concentration increases [68].
Additionally, the structural gallery of Ca v channels offers a glimpse into diverse conformations for the inactivated states.We propose classifying these states as "tight," "relaxed," and "loose" conformations to differentiate the structural variations within PD of the inactivated Ca v channels (Figure 3b).The "tight" PD, evident in the structures of Ca v 2.2 and Ca v 2.3 exhibiting voltage-dependent closed-state inactivation (CSI) [69], features a smaller central cavity volume with only a minor fenestration (Figure 3b, left).The intracellular gate is notably thick and tightened, facilitated by the presence of a Ca v 2-specific cytosolic helix at the II-III linker (designated as CH2 II ) [46][47][48].Supporting the structural analysis of CH2 II 's role in stabilizing the inactivated state, deletion of the CH2 II helix or mutation of the conserved Trp residue both exerted a substantial impact on channel inactivation, shifting the steadystate inactivation curves toward less negative voltages.Significantly, these mutations eliminated cumulative inactivation during action potential trains, indicating the crucial role of the CH2 II segment as a structural determinant for the CSI of Ca v 2 channels [47].The slightly "relaxed" PD, observed in most Ca v 1 and Ca v 3 channel structures, displays four fenestrations and a gate radius of approximately 1 Å (Figure 3b, middle).In these two types of conformations, the structure of SF remains relatively consistent.The "loose" PD configuration was recently identified in the structure of Ca v 1.2 in the presence of an atypical calcium antagonist [44].In this structure, the SF became dilated, and the gate, while not permeable, was sufficiently expanded to accommodate a detergent molecule (Figure 3b, right).

The structural arrangement of VSD
It was suggested that the conformational changes in VSDs in response to membrane depolarization were initially attributed to the movement of gating charge residues on S4 segments across the membrane electric field [23,70,71].As described in the sliding helix model, the transfer of gating charge residues through the hydrophobic constriction site is facilitated by the transient formation of ion pairs with countercharged residues on the opposite helices [71,72].As a result, the gating charge residues are thought to "slide" through the lipid bilayer, facilitating the conformational changes in the VSDs that ultimately lead to the opening or closing of the ion channel pore.
The resolved structures of VSDs can be categorized into two classes.In the structures of Ca  below (Figure 4a).In contrast, the structures of Ca v 2.2, Ca v 2.3 and Ca v 1.2 in the apo state reveal a down/deactivated state of VSD II , with the other three VSDs (VSD I , VSD III , and VSD IV ) remaining in the depolarized state (Figure 4b).In the down VSD II , the entire S4 segment transforms into a 3 10 helix, placing the gating charge residues on the same side.As shown in the structure of Ca v 2.2, only R2 is situated above the occluding Phe, while R3-K6 are situated below it.Upon superimposing the four VSDs, S1-S3 are relatively well aligned, whereas S4 II shifts significantly downward compared to the other S4 segments.
To identify the determinants governing the conformational transition of VSD II , we suggest comprehensive investigations that include constructing Ca v 1 and Ca v 2 chimeras and performing systematic mutagenesis analysis.The endeavors will significantly enhance our comprehension of the electromechanical coupling mechanism.

The molecular basis for Ca v channels modulated by endogenous components
The activity of Ca v channels is subject to modulations by a variety of endogenous components, such as calmodulin [73], G proteins [74], lipids [75], and synaptic associated proteins [76].Recent breakthroughs in elucidating the specific interactions involving calmodulin [77,78], components of muscle excitation-contraction coupling (STAC and junctophilin) [79,80], phosphatidylinositol 4,5-bisphosphate (PIP2) [46,48], as well as chaperones in the assembly of Ca v channels [81], have significantly deepened our understanding of Ca v channel modulations.
Calmodulin functions as the Ca 2+ sensor for calcium-dependent inactivation (CDI) of Ca v channels [82].We ever incubated human Ca v 3.1 protein with calmodulin, but the final cryo-EM map revealed the absence of calmodulin and a large portion of C-terminal domain (CTD) [50].Alternatively, insights into calmodulin-Ca v interactions were gained through X-ray and NMR structural analyses using only isoleucineglutamine (IQ) motif of Ca v .The IQ motif interacts with both N-lobe and C-lobe of Ca 2+ /calmodulin, with C-lobe showing higher affinity [77].
A recent NMR structure of Ca 2+ -free calmodulin (apo calmodulin) revealed that the IQ peptide interacted with the residues in C-lobe and adopted an orientation opposite to that in the structure with Ca 2+ /calmodulin (Figure 5a).The distinctive structural observations in Ca 2+ or Ca 2+ -free calmodulin may provide insights into CDI regulated by the Ca 2+ sensor protein [78].
Excitation-contraction coupling (ECC) in skeletal muscle necessitates functional and mechanical coordination between Ca v 1.1 and the ryanodine receptor (RyR1) [83,84].Recently, the Van Petegem group determined the structures of Ca v 1.1 peptides bound to STAC2 (Ca v 1.1 II-III loop) [80] and junctophilin 2 (Ca v 1.1 CTD) [79], revealing the interactions of Ca v 1.1 with the proposed auxiliary proteins involved in ECC.
PIP2 was initially observed to decelerate the "rundown" process and modify the voltage-dependence of Ca v 2.1 channels, a finding later corroborated in Ca v 2.2 channels [75,85,86].In the structure of Ca v 2.2 and Ca v 2.3 channels, PIP2 is coordinated at the interface between the down VSD II and PD (Figure 5b, left).The head group of PIP2 wedges into the cytosolic cavity within VSD II through the interplay of S3 and S4, while the hydrophobic residues on S3 to S6 in repeat II, as well as S5 and S6 in repeat III, coordinate the tails of PIP2.Gating charge residues R4 and K5 interact with the 5-phosphate group of PIP2 (Figure 5b, right).Moreover, the Ca v 2-specific CH2 II helix was noted to affect the coordination of PIP2 [48].In the CH2 II -deleted structure, the elevated shifts of S4-5 II and S5 II afford a more sharpened angle at the location where PIP2 was observed, rendering it incompatible for PIP2 binding.
The precise biogenesis processes play a pivotal role in ensuring the proper physiological functions of Ca v channels , [87].In eukaryotes, the conserved large nine-protein complex, termed endoplasmic reticulum membrane protein complex (EMC), has been shown to aid in efficient membrane insertion, and serve as a holdase for partially assembled membrane proteins [88].Various structural and mutagenesis studies have outlined the functional roles of the EMC and implied diverse interactions with client proteins [89][90] [91].The recent structure of Ca v channel bound to EMC revealed extensive interactions at transmembrane and cytosolic docking sites (Figure 5c) [43].Guided by the structural analysis, mutagenesis at the EMC-Ca v interface, particularly at the Ca v β-Cyto dock, resulted in a substantial reduction in Ca v channel currents.The structure-function analysis underscores the role of EMC as a channel holdase to facilitate the assembly and functional expression of Ca v channels, thereby advancing our comprehension of the ion channel assembly intermediate.

Structural pharmacology of Ca v channels
In this section, we will present an overview of the recent advancements in the structural pharmacology of Ca v channels.The structural gallery, revealing the diverse MOAs of small molecule drugs and peptide toxins, is expected to greatly accelerate the future drug discovery efforts targeting Ca v channelopathies.

Structural pharmacology of DHP drugs
DHP drugs, such as nifedipine, and amlodipine, have been extensively utilized in clinical settings for the effective management of hypertension.By inhibiting LTCCs in cardiovascular tissue cells, DHP drugs reduce peripheral vascular resistance and lower blood pressure [16,92,93].The molecular mechanisms underlying the modulation of Ca v channels by representative DHP drugs, including nifedipine, amlodipine, (R)-(+)-Bay K8644 and (S)-(-)-Bay K8644, have been elucidated using rabbit Ca v 1.1 as the prototype [39,40].The DHP drugs bind to the fenestration site enclosed by the pore-forming segments in Repeat III and IV (Figure 6a, DHP).Consequently, these drugs regulate Ca v channels through an allosteric modulation mechanism instead of directly obstructing the ion-permeation path.The conserved N1 amine of the DHP core plays a crucial role by forming a hydrogen bond with surrounding residues [94,95].Furthermore, the two oxygens of the C3-ester group, which appear to be crucial for antagonistic activities, are also H-bonded to the polar residues conserved in the Ca v 1 subfamily [96,97].
Among DHP drugs, amlodipine demonstrates remarkable potency and a pH-dependent inhibitory impact [98].The ethylamine side chain has been noted to extend toward the ion pore, interacting with a phospholipid molecule that transverses the central cavity.This unique MOA may distinguish the efficacy of amlodipine from that of other DHP drugs [40].

Structural pharmacology of small molecule pore blockers
In addition to DHP drugs, a variety of small molecule drugs, exemplified by diltiazem and verapamil, are commonly used to manage cardiovascular conditions.Unlike DHP compounds, these drugs inhibit Ca v activities by directly obstructing the ion-permeation path, classifying them as pore blockers.While the pore blockers accommodate into the proximal central cavity pockets, there are slight differences in the coordination details [39,45,47,51].
Diltiazem and verapamil serve as the prototypes for benzothiazepine and phenylalkylamine drugs, respectively, which are commonly used to treat hypertension and cardiac arrhythmias.In the resolved structures, diltiazem predominantly engages with numerous hydrophobic residues on S6 I , P1 III , S6 III , and S6 IV (Figure 6a, Diltiazem) [39].In contrast, verapamil potentially adopts two distinct binding modes [39].In the first mode, verapamil is primarily coordinated by the hydrophobic residues on P1 II , S6 II and S6 III .The second mode, where verapamil interacts mainly with the hydrophobic residues on S6 IV , aligns with the previous biophysical characterizations [99].
Cinnarizine functions as a nonselective Ca v blocker employed in the treatment of motion sickness [100].It was observed that cinnarizine binds within the central cavity of PD, with the styrene group interacting with the residues on SF vestibule [45].The core scaffold, diphenylmethylpiperazine group, fits into the cavity formed by the hydrophobic residues on repeat II and III, thereby stabilizing the overall binding of the molecule.Beside steric blockage, local structural shifts resulting from cinnarizine binding induce an α→π transition of the helical turn constituting the intracellular gate, further constricting the ion-permeation pore.
The structural basis for pore blockage of LVA channels, T-type Ca v channels, has also been elucidated [50,51].Z944 exhibits characteristics of both a pore blocker and an allosteric antagonist [50].In the high-resolution structure, Z944 interacts with the II-III fenestration rather than the typical DHP binding pocket at the III-IV fenestration [39].The phenyl ring is positioned into the fenestration site, while the tert-butyl group is directed toward the ion pore, impeding the permeation of ions.Similar to the cinnarizine-bound structure, Z944 binding induces an α→π transition in S6 II .A recent study has further unveiled analogous binding modes observed in Ca v 3.3 channels complexed with mibefradil, pimozide, and otilonium bromide [51].
Two inhibitors, PD17312 and Ca v 2.2 blocker 1, were observed to block Ca v 2.2 channels through a dual mechanism involving both pore blockage and allosteric modulation [47].Despite having distinct chemical structures, the two molecules occupy the same binding site (III-IV fenestration) within Ca v 2.2.Unveiling the structural details of the III-IV fenestration site will facilitate the drug discovery efforts aimed at targeting Ca v 2.2 for the treatment of chronic pain.[39]; Diltiazem: interacts with the hydrophobic residues on S6 I , P1 III , S6 III , and S6 IV .Replacement of

Structural basis for the adverse interactions of sofosbuvir and amiodarone on LTCCs
Sofosbuvir, which targets NS5B polymerase of the hepatitis C virus, has achieved almost 100% cure rates for hepatitis C [101][102][103][104]. Nevertheless, co-administration with amiodarone, an antiarrhythmic drug that primarily inhibits diverse ion channels in heart, leads to a drug-drug interaction causing severe bradycardia [105][106][107].Analysis of the cryo-EM structures of LTCCs in the presence of the two drugs uncovered a direct drug-drug interaction within the channels, shedding light on the concerning clinical observation [41].
Amiodarone binds to the III-IV fenestration of the LTCC through extensive hydrophobic interactions (Figure 6a, Drug-drug interaction).However, when administered alone, sofosbuvir (or its analogue MNI-1) is not present in the structure.The synergistic inhibition of Ca v channels by amiodarone and sofosbuvir (or its analogue MNI-1) is clearly elucidated by the two drug-bound structures.Amiodarone anchors sofosbuvir/MNI-1 to the central cavity of LTCC, effectively obstructing the ion-conducting pathway.The drug-drug interaction is facilitated by the hydrophobic contacts and notably enhanced by a polar interaction between the phosphate group of sofosbuvir/MNI-1 and the tertiary amine of amiodarone.Observing the direct physical and pharmacodynamic interaction of amiodarone and sofosbuvir/MNI-1 on the scaffold of LTCCs underscores the clinically pertinent, potentially life-threatening nature of this drug-drug interaction [41].
The molecular recognition of the gabapentinoid drugs, gabapentin and mirogabalin, by the α2δ-1 subunit have been investigated, but the structural observations are somewhat ambiguous [42,52].Gabapentinoid drugs were supposed to occupy a pocket identical to the L-leucine binding site at the Cache1 domain (Figure 2B).No discernible conformational change was observed in the α2δ-1 subunit upon binding to gabapentin or mirogabalin.However, our group ever refrained from conclusively assigning densities in the same site to gabapentinoid (pregabalin) due to its similar size and shape to the endogenous L-leucine ligand [27].It remains to be investigated how gabapentinoids regulate the function of neuronal Ca v channels if there is no conformational alternation for the α2δ-1 subunit.

Structural basis for Ca v channels blocked by peptide toxins
Peptide toxins have historically been valuable tools for understanding the physiological functions of ion channels.Moreover, ongoing advancements Tyr1365, Ala1369, and Ile1372 on S6 IV with non-LTCC residues was shown to significantly decrease the sensitivity to the drug [39]; Drug-drug interaction: AMIO (chocolate ball-and-stick) binds within the III-IV fenestration and mainly interacts with the hydrophobic residues on S6 III and S6 IV .AMIO anchors the accommodation of SOF (pink ball-and-stick) into the cental cavity through hydrophobic contacts (black dashes) and a polar interaction (cyan dashes) between the phosphate group of SOF and the tertiary amine of AMIO [41]; Ziconotide (chocolate): Arg10 and Tyr13 of ziconotide are H-bonded to Asp664 on P2 II , Ser19 is H-bonded to Glu1659 on P2 IV , Thr17 is H-bonded with Asp1345 on ECL III , Arg21 and Lys4 interact with Asp1628 and Asp1629 on ECL IV , respectively.Four of the eight ziconotide-coordinating residues in Ca v 2.2, Thr643, Asp1345, Lys1372 and Asp1629 (blue label), are not conserved in other Ca v channels [46]; Calciseptine (light purple): Arg31 and Gln49 of CaS are H-bonded to Trp1111 (O) and Asn1113 (N) on ECL III , Arg28 is H-bonded to Ser1114 and Asp1117 on ECL III , Gln30 interacts with Gly1476 (O), Trp46 is surrounded by hydrophobic residues Phe1116 on ECL III , Met1126 on P2 III , and Leu1471 on P2 IV .Replacement of Ca v 1.2-specific residues at the channel-toxin interface with their counterparts in Ca v 1.1 or Ca v 2.2 (D1117H, V1501Y, N1113K, and A1123W) exhibited reduced sensitivity to calciseptine, revealing the molecular basis for the subtype specificity [44].(b) the diverse configurations of Ca v channels in complex with various drugs and toxins.Pinaverium bromide (PIN) captures Ca v 1.2 in an inactivated state with a loose PD.Upon PIN binding, the transverse S4-5 III helix combines with the S5 III segment to form a single straight helix (orange arrow).P2 III helix is missing in the cryo-EM map (yellow dashes).The dilated intracellular gate is fitted with a GDN molecule.Shown below is a schematic cartoon illustrating different conformations for the inactivated states.The drugs and toxins that prefer "tight," "relaxed" and "loose" PD, as revealed by cryo-EM structures, are indicated.The binding details for ziconotide in panle A, and panel B were adapted from our published paper [44,46] with modifications.
in peptide chemistry, drug delivery, and formulation are making peptide toxins an increasingly attractive avenue for the development of ion channel drugs [114].
Ziconotide, derived from ω-conotoxin MVIIA, acts as a selective blocker for Ca v 2.2 channels and received FDA approval in 2004 for the treatment of severe chronic pain [18,19,115].Structural studies have unveiled the molecular underpinnings of the specific pore blockage of ziconotide on human Ca v 2.2 [46,47].Ziconotide is situated in the electronegative pocket surrounding the entrance to SF (Figure 6a, Ziconotide).To accommodate ziconotide, ECL III must move upward together with the α2δ-1 subunit.The positively charged residues in ziconotide effectively neutralize the negatively charged pocket that attracts ions into the ion entrance, creating a spatial obstruction to prevent Ca 2+ influx into the pore.Further sequence comparison revealed that half of the crucial residues required for ziconotide coordination were not conserved among Ca v channels, thus elucidating the molecular basis for the selective pore blockade of ziconotide.
Calciseptine, a member of the three-finger toxin isolated from the venom of black mamba, consists of 60 amino acids and features four pairs of disulfide bonds.With the ability to selectively block Ca v 1.2 and Ca v 1.3 channels, calciseptine effectively inhibits the contraction of smooth and cardiac muscles [116][117][118][119][120][121].In contrast to other VSD or PD-binding toxins, calciseptine is positioned on the PD shoulder and thoroughly interacts with the P2 pore helices and the ECLs in repeats III and IV (Figure 6a, Calciseptine) [44].Mutation assays have highlighted the role of Ca v 1.2-specific residues, mapped at the protein-toxin interface, in determining the subtype specificity.The unexpected binding mode of calciseptine provides a valuable template and induces novel concepts for the development of innovative antihypertensive medications.

Pinaverium bromide traps Ca v 1.2 in an inactivated state with a loose PD
Studies in structural pharmacology not only clarify the MOAs of various drugs, but also have the potential to capture additional functional states of Ca v channels.However, most investigations have yielded the conformations similar to the apo state.For instance, the structures of Ca v .2.2/Ca v 2.3 in the apo state or in complex with drugs often exhibit an inactivated state with a tight PD (Figure 6b, left).Additionally, the structures of most drug bound LTCCs show a relaxed PD, similar to the apo structure (Figure 6b, middle).Our group recently revealed that pinaverium bromide (PIN), an antispasmodic agent used to alleviate symptoms of irritable bowel syndrome, could trap Ca v 1.2 in a conformation distinct to the apo state [44].
PIN potentially wedges into the corner enclosed by S4-5 III , S6 III and S6 IV .Upon binding to PIN, Ca v 1.2 undergoes a substantial range of conformational changes, including a structural transition of VSD II from a down to an up state, a significant reshaping of SF vestibule, and dilation of intracellular gate (Figure 6b, right, Loose PD).Despite the considerable expansion of the ion-permeation path, molecular dynamics simulations reveal that the PIN-bound state remains non-conductive for Ca 2+ ions, thereby supporting the antagonistic activities of PIN for Ca v 1.2.

Conclusion
Ca v channels are of great physiological importance across cardiovascular, neuronal, and endocrine systems, serving as primary targets for treating diseases such as hypertension, cardiac arrhythmia, chronic pain, and epilepsy.In this review, we have summarized the strides made and novel insights achieved through the structural analyses of different Ca v subtypes.The structure gallery establishes the foundation for dissecting the physiological functions of Ca v channels and underlying the electromechanical coupling mechanisms.Moreover, the elucidation of structures in complex with clinically relevant drugs and promising ligands offers detailed insights into the structural pharmacology of Ca v channels.These studies advance our comprehension of the working mechanisms, and pave the way for prospective drug discovery against Ca v channelopathies.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was not supported by any funding.

Figure 1 .
Figure 1.Classification, pharmacology, and topology of Ca v channels.(a) the ten primary subtypes of Ca v channels are classified into three groups-Ca v 1, Ca v 2, and Ca v 3-based on the sequence similarities of the α1 subunits.The Ca v 1 and Ca v 2 channels belong to high-voltage activated (HVA) channels, while Ca v 3 members are low-voltage activated (LVA) channels.Also shown are the genes that encode the α1 subunits and the current type characterized by electrophysiological experiments.The representative drugs of each isoform are listed.(b) chemical structures of small molecule drugs whose MOAs have been determined by high-resolution structures.Please refer to table 1 for PDB codes and additional details.(c) the HVA channels consist of at least three subunits: the transmembrane α1 subunit, the extracellular α2δ subunit, and the cytosolic β subunit.The α1 subunit comprises four homologous

Figure 2 .
Figure 2. The subunit architecture of Ca v channels.(a) the overall structures of rabbit Ca v 1.1 (PDB: 5GJV), human Ca v 2.2 (PDB: 7MIY) and human Ca v 3.1 (PDB: 6KZO).All structures are domain-colored.In Ca v 2.2, the β3 subunit is sandwiched between AID and the S6 II cytosolic segment (S6 II C).The Ca v 2-specifc cytosolic helix in repeat II (CH2 II ) is also labeled.The lipid molecule, phosphatidylinositol 4,5-bisphosphate (PIP2, black stick), is coordinated with the interface between down VSD II and PD.CTD, C-terminal domain (forest cartoon).ECL, extracellular loop.(b) structural topology of the α2δ-1 subunit.The VWA domain and four cache domains (domaincolored) are intertwined in the primary sequence.The α1 subunit interacts with VWA (palecyan), Cache1 (light pink) and the intervening loop of the Cache2 domain (light blue) of the α2δ-1 subunit.The interface between VWA and α1 subunit involves the coordination of a Ca 2+ ion (green sphere), facilitated by the metal ion-dependent adhesion site (MIDAS) site and the Asp residue on the S1-2 loop of repeat I.

Figure 3 .
Figure 3.The structures of PD.(a) the putative Ca 2+ coordination in SF vestibule.Ca 2+ ions are coordinated by the EEEE residues for Ca v 1 and Ca v 2 channels, contrasting with EEDD residues in Ca v 3 channels.The distinct features of Ca 2+ ion densities (purple and brown meshes) are observed at concentrations of 0.5 and 10 mM, respectively.(b) the resolved structures of PD can be classified into three conformations, tight PD (represented by Ca v 2.2, PDB: 7MIY), relaxed PD (represented by Ca v 1.1, PDB: 5GJV), and loose PD (represented by Ca v 1.2, PDB: 8WEA).The variations include the volume of the central cavity, the composition and size of the intracellular gate, and the contour of SF.The α1 structures are domain colored (grey for repeat I, cyan for repeat II, yellow for repeat III, and palegreen for repeat IV).The Ca v 2-specific CH2 II segment is shown as orange cartoon, with the conserved Trp residue shown as orange ball-and-stick.The hydrophobic residues constituting intracellular gate are depicted as sticks.The detergent molecule, glyco-diosgenin (GDN), is nestle into the expanded intracellular gate, as seen in the loose PD.Panel a was adapted from our published papers [27,50] with minor modifications.
v 1.1, Ca v 1.3, and Ca v 3 channels, all four VSDs are characterized as depolarized or up state.As depicted in the structure of Ca v 1.1, the gating charge residues R1-R4 within the four VSDs are positioned above the conserved occluding Phe in the charge transfer center.Meanwhile, R5 and R6 are positioned

Figure 4 .
Figure 4.The structures of VSDs.(a) the depolarized conformations of the four VSDs, as seen in the Ca v 1.1 structure (PDB: 5GJV).The gating charge residues on S4, An1 and An2 (conserved acidic or polar residues on S2), as well as the occluding Phe, are shown as sticks.The gating charge residues above and below the occluding Phe are labelled cyan and brown, respectively.In the depolarized conformation, R1-R4 are positioned above the occluding Phe.(b) the structure of the four VSDs in human Ca v 2.2 (PDB: 7MIX).VSD II (brown labeled) is in a down/deactivated conformation, while the other three VSDs remain in the depolarized state.In the down VSD II , the gating charge residues R3-K6 are below the occluding Phe (brown labeled), with only R2 above it (cyan labeled).K6 of VSD II is on the opposite side to the other four gating charge residues and projects into the cytosol.Panel b was adapted from our published paper[46] with minor modifications.

Figure 5 .
Figure 5. Structural basis for Ca v channels modulated by endogenous components.(a) different binding modes of the Ca v IQ motif (cyan and orange cartoon) with Ca 2+ /calmodulin (palegreen, PDB: 2BE6) or with apo calmodulin (wheat, PDB: 6CTB).Green spheres indicate Ca 2+ ions.(b) binding details of PIP 2 in the Ca v 2.2 structure (PDB: 7MIY).Left: the PIP2 molecule is coordinated at the interface between down VSD II and PD.right: the PIP2 molecule is coordinated with the polar residues in Ca v 2.2.Hydrogen bond interactions are shown as blue dashes.(c) structure of the EMC chaperone-Ca v assembly intermediate.The EMC -Ca v is an approximately 0.6 MDa complex with dimensions of about 220 Å normal to the membrane plane and around 100 Å × 130 Å parallel to the membrane plane (PDB: 8EOI).EMC1-8 and 10, and the α1 and β3 subunits of Ca v 1.2 are shown.The EMC extensively interacts with the α1 and β3 at the transmembrane (TM dock) and cytosolic regions (cyto dock).

Figure 6 .
Figure 6.Overview of the structural pharmacology of Ca v channels.(a) structural mapping of the drug binding sites in the Ca v structures.Representative DHP drug (nifedipine, black ball-and-stick) and pore blocker (Diltiazem, magenta ball-and-stick) are shown.key interactions of representative drugs are summarized below.Hydrogen bonds are indicated by red dashes.Nifedipine:the N1 amine is hydrogen-bonded (H-bonded) to the hydroxyl group of Ser1011 on P1 III , the two oxygen groups of the C3-ester are each H-bonded to Thr935 and Gln939 on S5 III , the nitrophenyl ring is situated within a hydrophobic pocket formed by Val932 on S5 III as well as Met1057 and Phe1060 on S6 III , the DHP backbone and the methyl groups are surrounded by Phe1008 on P1 III as well as Tyr1365 and Met1366 on S6 IV[39]; Diltiazem: interacts with the hydrophobic residues on S6 I , P1 III , S6 III , and S6 IV .Replacement of

Table 1 .
Published cryo-EM structures of Ca v channels.
observed in the structures, the VWA, Cache1, and the intervening loop of Cache2 domains are prominently engaged in interactions with the α1 subunit, particularly with the extracellular loops of repeats I to III (ECL I to ECL III ) and the intervening loop between S1 and S2 of repeat I (S1-2 I